PROJECT SUMMARY Biliary atresia (BA) is a fibro-obliterative disease of the extrahepatic bile ducts (EHBDs) affecting neonates around the world. BA is the most common indication for liver transplant in the pediatric population, with 50% of patients requiring transplant by age 2 and most of the rest before adulthood. The etiology and early course of the disease are unknown, but recent data suggest that BA results from a prenatal environmental insult in the setting of genetic susceptibility, followed by progression of the original injury. We identified and synthesized a previously unknown isoflavonoid, biliatresone, that is found in Australian plants and is likely responsible for four large outbreaks of BA in Australian livestock. This toxin causes a BA- like disease in larval zebrafish, rapid increases in the permeability of the monolayer in cholangiocyte organoids, and, remarkably, obstruction and peri-ductal fibrosis in neonatal mouse EHBD explants. We have now developed a powerful new animal model whereby toxin treatment of pregnant mice causes EHBD damage in pups, and progression is secondary to harmful bile acids. This proposal seeks to use biliatresone and other model systems to answer three key questions in BA: 1) Why is toxicity specific to neonates? 2) What determines repair vs. progression of cholangiocyte injury? and 3) How and why does fibrosis occur in the EHBD? Our underlying hypothesis is that the answers to these questions are found in the unique features of the neonatal biliary system and that BA results from the combination of duct susceptibility, a primary injury, and injury progression and fibrosis secondary to bile acids and the unique anatomic features of the neonatal duct. Our preliminary data identify five key features of the neonatal bile ducts that potentially increase their susceptibility to injury. We show that neonatal EHBDs lack a protective glycocalyx, have immature cell-cell junctions, and have relatively low levels of GSH compared to the liver. We also show that the submucosa of the neonatal EHBD contains a large number of likely fibrogenic cells that may be ?primed' to respond to insults and that the anatomical structure of the neonatal submucosa may propagate injury. Based on these preliminary data, we propose that BA results from a ?second hit? in the context of a susceptible duct. We have organized our hypotheses and aims around three key questions in BA. Specifically, we address why injury is specific to neonates, why it preferentially affects the EHBD, and how fibrosis develops. Our three specific aims focus on 1) neonatal susceptibility to injury, including the role of the glycocalyx; 2) the determinants of injury progression, including the role of the bile acids; and 3) the identity and activating stimuli for the fibrogenic cells of the EHBD. We will use a combination of cell-based and mouse experiments to achieve these aims. This work has the potential to significantly shift our understanding of BA and lead to new therapeutic options in a disease that almost inevitably leads to liver transplant.